Allergy is a serious medical condition affecting millions of people worldwide, with about 15 million people in the United States, including many children. During an allergic reaction, the immune system mistakenly targets an allergen as a threat and attacks it. The allergic reaction may affect the skin, the digestive system, the gastrointestinal tract, the respiratory system, the circulatory system and the cardiovascular system; in some allergic reactions, multiple organ systems are affected. Allergic reactions range from mild to severe or life-threatening. Severe symptoms may include difficulty in breathing, low blood pressure, chest pain, loss of consciousness, and anaphylaxis. People having allergies currently manage their allergies by avoiding any food that might contain that specific allergen. These restrictions have a major impact on the patients' quality of life and there remains no method for assessing the true allergen content of food. In the United States, food allergy symptoms send someone to the emergency room every three minutes. A rapid method for determining the presence of an allergen would be of great benefit. A portable device that enables the patients to test their food and determine accurately and immediately the allergen content will be beneficial to provide for an informed decision on whether to consume or not.
U.S. Pat. No. 5,824,554 to McKay teaches a dining mat formed of an absorbent material and small spots of chemical reagents applied to isolated zones on the mat, for detection of food allergens. If the food product contains the allergenic substance, the chemical reagent will change its appearance indicating the presence of the allergenic substance in the food product. The detection limit and the detection specificity are limited by the chemical reagent used in the spots. A drawback is that false negatives are highly possible when analyzing solid food products because of the long reaction times between the solid food products and the spot reagent.
US Patent Application Pub. No. 2008/0182339 and U.S. Pat. No. 8,617,903 to Jung et al. teaches a method of detecting an allergen by processing samples with microfluidic chips configured for analysis of one or more allergen indicators, detecting the allergen indicators with one or more detection units, and displaying results with one or more display units. The detecting system comprises a microfluidic chip, a reagent delivery unit, a centrifugation unit, an analysis unit, a detection unit, a display unit, and a recording unit. The device is not sufficiently compact to be portable.
US Patent Application Pub. No. 2010/0210033 to Scott et al. teaches a portable device for detecting food allergens comprising a housing, a sample inlet port, a means for indicating the presence of the potential allergen in the sample, and an allergen detection chip comprising an antibody to the potential allergen, wherein the antibody is labeled with a detectable tag.
U.S. Pat. No. 7,527,765 to Royds teaches a food testing device for identifying the presence of harmful contaminants in a food sample, comprising a disposable sample container, a mechanical liquefier including a blade assembly, a test supply compartment with a reagent having an affinity for the harmful contaminant and capable of detecting the harmful contaminant in the liquefied food sample, and producing a visual cue upon recognition of the harmful contaminant.
Aptamers, as well as devices and methods of using them in the detection of proteins in food, are disclosed in several patents and patent applications (each of which is incorporated herein by reference in its entirety), including: U.S. Pat. No. 8,633,140 to Kim, et al., which teaches a microarray of functionalized polydiacetylene molecular sensors; U.S. Pat. No. 8,618,046 to Brunner, et al., which teaches a method for treating atherosclerosis using aptamer-based anti-CETP-antibody-inducing antigens; and U.S. Pat. No. 8,614,466 to Rasooly, et al., which teaches a method and system employing a physical principle called “electrical percolation,” (flow of electricity through a random resistive network) for electrically detecting biomolecular binding in a semiconductor. In one embodiment, capture molecules for binding target molecules can be an aptamer. U.S. Pat. No. 8,563,298 to Lowery, Jr., et al. teaches NMR systems and methods for the collection and detection of analytes. U.S. Pat. No. 8,507,458 to Yokota, et al. teaches a system for delivering nucleic acids for suppressing target gene expression by utilizing endogenous chylomicron, wherein the nucleic acid may be an aptamer. U.S. Pat. No. 8,236,933 to Herzog, et al. teaches transgenic animals having a reduced level of expression of peptide YY (PYY) and methods of using the transgenic animals for screening a library of aptamers and identifying agonists and antagonists of PYY. U.S. Pat. No. 8,232,584 to Lieber, et al. teaches a fluorescence based nanoscale wire biosensor devices and methods for detecting analytes, wherein an aptamer may be indirectly immobilized relative to the nanoscale wire. U.S. Pat. No. 7,977,462 to Hornbeck et al. teaches lateral flow devices for detecting and quantitating novel tyrosine phosphorylation sites identified in carcinoma and/or leukemia. U.S. Pat. No. 7,973,079 to Mata, et al. teaches biosensors for detecting macromolecules and other analytes that can modulate the activity or availability of serum retinol, retinol-binding protein (RBP) and/or transthyretin (TTR). U.S. Pat. No. 7,855,057 to Gordon, et al. teaches methods, reagents and apparati for detecting small quantities of protein isoforms (e.g., due to alternative splicing, or different disease protein isoforms or degradation products) in a sample, including using combinations of capture agents, wherein the capture agent may be an aptamer. U.S. Pat. No. 7,850,964 to Vukicevic, et al. teaches nucleic acid biosensors of bone morphogenetic proteins (BMPs), e.g., BMP-1 procollagen c-proteinase, for diagnosis and treatment of bone and soft tissue defects and disorders.
Anaphylatoxin C5a- (complement factor 5a)-binding aptamers are described in PCT Publications WO 2009/040113, WO 2010/108657 and WO 2013/104540 to Buchner, et al. Buchner, et al. also describe aptamers that bind to CXC chemokine stromal cell-derived factor-1 (SDF-I) in PCT Publication WO 2009/019007.
Molecular beacons (MBs) are hairpin-shaped oligonucleotides that contain both fluorophore and quencher moieties and act like switches. When in a closed state, the fluorophore and quencher are brought together and the fluorescence is quenched (“turned off”) by resonance energy transfer. When a conformational change opens the hairpin structure and the fluorophore and quencher are separated, the quencher can no longer quench and fluorescence is restored (“turned on”). MBs are particularly useful in detection devices and diagnostic assays requiring a probe to have high sensitivity and excellent molecular recognition specificity; they are extraordinarily target-specific, ignoring nucleic acid target sequences that differ by as little as a single nucleotide. Other advantages of MBs are: (1) sensitivity can allow for real-time monitoring; (2) low background signal allows for a fluorescence enhancement of more than 200 times; (3) MBs allow “detection without separation,” where it is impossible or undesirable to isolate the probe-target hybrids from an excess of the unhybridized probes. The specificity provided by the MB loop-stem structure has been demonstrated to be applicable in a variety of biological environments. The compositions, methods and devices disclosed herein are applicable in solution-based (in vitro) investigations of RNA-DNA interactions, protein-DNA interaction studies, measurements within living systems, and biosensor design. For example, compositions described herein can be used in in vitro investigations such as real-time monitoring of DNA/RNA amplification during PCR; rapid and reliable mutation detection for clinical diagnosis (Xiao, et al., (2009) Fluorescence Detection of Single Nucleotide Polymorphisms via a Single, Self-Complementary, Triple-stem DNA Probe. Angew Chem. Int. Ed. Engl. 48(24):4354-4358); spectral genotyping (Kostrikis, et al., Science, 1998, 279: 1228); DNA sticky-end pairing (SEP) analysis; visualization of subcellular localization and cellular transport pathway of RNAs (Tan et al., (2005) Molecular Beacons for Bioanalytical Applications. Analyst 130: 1002-1005).
Exemplary molecular beacons are reviewed in Leung, et al., 2011 (Nucleic Acids Research, 2012, 40(3):941-955) and described in U.S. Pat. No. 8,188,255 to Litman et al., which teaches microRNA (miRNA) sequences associated with cancer, and their detection using aptamers and molecular beacons; U.S. Pat. No. 7,282,360 to Meyers et al., which teaches novel protein kinase, serine/threonine protein kinase, serine/threonine phosphatase, prolyl oligopeptidase, trypsin, serine protease, and ubiquitin carboxy-terminal hydrolase family members, referred to herein as “53070, 15985, 26583, 21953, m32404, 14089, and 23436,” and generally discloses detection of them using aptamers or molecular beacons; and U.S. Pat. No. 6,730,491 to Kapeller-Libermann et al., which teaches three allegedly novel protein kinase family members, referred to herein as “2504, 15977, and 14760” and generally discloses detection of them using aptamers or molecular beacons.
There remains a need for a portable and reusable device for fast and accurate detecting allergens. There also remains a need for detecting multiple allergens with a single device.